In an effort to define immunodominant epitopes and to develop a specific skin test reagent that will distinguish Mycobacterium tuberculosis infections from those caused by the Mycobacterium avium, Mycobacterium intracellulare complex (MAC), T cell epitopes from mycobacterial antigens are being defined. Peptides deduced from both published mycobacterial antigen DNA sequences and DNA sequences determined in the Laboratory of Immunology are being synthesized, chemically modified for testing, and prepared for possible inclusion in multiple epitope reagents. For testing, these small peptides (1200-2000 kDa) are polymerized by means of chloroacetyl alkylating group which is added to the N-terminus of each peptide. Three variations of the method have been developed for use with peptides of different compositions. The above methods are also being adapted for use in the preparation of multiple epitope reagents. T cell activity is being tested by skin tests and by T cell stimulation in vitro. T cell lines against M. avium, M. intracellulare, M. terrae, M. scrofulaceum, M. fortuitum, M. kansasii, M. tuberculosis, and M. bovis (BCG) have been developed. The sequences of homologous 19 kDa antigens from M. tuberculosis and M. intracellulare are currently under investigation by synthesis and analysis of peptides deduced from the DNA sequences. The two antigens, exhibiting 78% homology, present an excellent opportunity for the definition of epitopes specific for one bacterium or the other. Twenty-six peptides, covering all of the residues differing in the two proteins, have been synthesized, purified, and polymerized. Both in vivo and in vitro screening for T cell epitopes have been initiated. Initial studies have identified several peptides which stimulate in vitro and in vivo cellular responses.